The obligate human parasite N. gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhea. The gonococcal pilus is an essential virulence factor required for infection of human volunteers. The amino acid sequence of the major subunit of the pilus, pilin, can rapidly change of many silent pilin loci to the pilin expression locus by nonreciprocal, homologous recombination. There has been shown in vitro to result form autolysis and transformation of silent sequences into the expressed gene. It has also been proposed that an intracellular recombination system may operate to promote nonreciprocal recombination between the pilin silent and expression loci (gene conversion). We are investigating the specific molecular mechanisms that produce pilin antigenic variation. We have shown that efficient antigenic variation requires a conserved DNA sequence, the Sma/Cla repeat, which is found in every pilin locus and only in pilin loci. This repeat shows high sequence similarity to known site-specific recombination enzyme binding sites, with two sets of binding sequences contained within the repeat. Consistent with this observation, several proteins have been detected that specifically bind the repeat. Mapping the functional sequences of the repeat will determine how many binding sequences are contained in the repeat. Isolating and characterizing the enzymatic activities that act at the site will allow us to understand how the Sma/Cla-specific system is involved in promoting homologous recombination to produce an antigenically variant pilus. In addition, a series of genetic constructs has been generated to provide strains to test if variable pilin sequences transfer more efficiently within a cell (gene conversion) or between cells (autolysis/transformation). Co-culture experiments will also be used to determine if the components of the Sma/Cla-dependent system act within a single cell or between cells. This planned studies will not only refine our models of how gene translocation are produced in the gonococcus, but will also contribute to understanding the mechanisms used to promote high frequency gene translocation in other systems. Moreover, understanding the genetic capabilities of this microbe is essential to understanding the pathogenesis of gonorrhea, since it is the physiological capabilities of this organism that allow it to grow in vivo, evade immune responses, adapt to new environments, and transfer to new hosts. The information gained in these studies will allow a better comprehension of the genetic mechanisms used by this bacterium to promote human disease.